JP2860963B2 - New substance SBS and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel substance SBS having a platelet aggregation inhibitory action and an antitumor promoter action, and a method for producing the same.

[Conventional technology]

In recent years, bioactive substances from basidiomycetes have been actively searched for, and various reports have been made. For example, antitumor substances have been found from Kawatake mushroom and Suehirotake mushroom, blood pressure lowering substances have been found from Mannake mushroom, and immunostimulants, antiallergic substances, antiulcer substances and the like have been found from various basidiomycetes. Pharmacological effects have also been found in mushrooms that are commonly eaten, and antitumor substances are more effective than shiitake mushrooms and enokitake and blood pressure lowering effects than oyster mushrooms (Japanese Unexamined Patent Publication No.
-72121). In addition, a special research conducted by the Ministry of Education, “Systematic Analysis and Development of Food Functions,” has triggered the attention of functional foods that claim to be tertiary functions of foods. Searches for physiologically active substances in foods have also been actively conducted.

[Problems to be solved by the invention]

An object of the present invention is to provide a novel physiologically active substance produced by Liophyllum ulmarium, which is eaten as a food mushroom along with Shiitake mushroom and Enokitake mushroom, and a method for producing the same.

[Means for solving the problem]

In summary, the first invention of the present invention is a novel substance
SBS is characterized by having the following physicochemical substances.

Elemental analysis: C 73.06% H 11.85% Molecular weight: 738 (according to FAB-mass spectrum) Molecular formula: C ₄₅ H ₈₆ O ₇ ultraviolet absorption spectrum (in methanol): shows a maximum absorption at 208 nm as shown in FIG.

Infrared absorption spectrum (KBr tablet): As shown in FIG. 2, absorption is shown at 3400, 2990, 2960, 2920, 1380, 1180, and 945 cm ⁻¹ .

Color reaction: negative for ninhydrin reaction, negative for phenol sulfate reaction, positive for iodine reaction, positive for decolorization reaction of potassium permanganate Further, the second invention of the present invention relates to a method for producing a novel substance SBS, and a novel substance SBS belonging to the genus Liofiram. The novel substance SBS of the first invention is collected from a culture of a producing bacterium or a fruit body of the producing bacterium.

 As an example of the present invention, the fruiting body of a novel substance SBS-producing bacterium
As for the product name "Yamabiko "
Artificially cultivated and commercially available can be used. Also fungus
Examples of strain cultures include Liofiram ulmarium
A culture of Lu1-8 (FERM BP-1416) is available.
The microbiological properties of the strain are described in JP-A-63-273467.
The novel substance used in the present invention SBS-producing bacteria
Is a strain belonging to the genus Liophyllam, not limited to the above strains.
Any strain can be used.

The method for producing the novel substance SBS comprises three steps of obtaining, extracting and purifying fruit bodies or cultured cells. The fruiting body is easily available as a commercial product, and the cultured cells are obtained by a general liquid culture method. For example, glucose, sucrose, starch, dextrin, glycerin, organic carbons such as fats and oils that can be used as a carbon source, yeast extract, peptone, corn steep liquor, defatted soybean, soybean hull, meat extract, amino acid, ammonium salt as a nitrogen source Organic nitrogen compounds and inorganic nitrogen compounds can be used. Besides that,
Inorganic salts such as magnesium salts, phosphates and potassium salts may be added. The culture temperature is suitably 15-30 ° C,
~ 25 ° C is preferred. The culture pH is suitably from 5 to 9;
~ 8 is preferred. The culture may be either shaking culture or static culture, and cells can be obtained in 5 to 15 days. However, static culture produces higher SBS. Next, extraction is performed using an organic solvent such as methanol, ethanol, acetone, ethyl acetate, and chloroform. For purification, general separation and purification methods such as adsorption chromatography of normal-phase and reverse-phase silica gel, reverse-phase ODS, cellulose and the like, gel filtration, and a method utilizing a difference in solubility in various solvents can be used.

The novel substance SBS obtained as described above has the following physicochemical properties.

1. Appearance: white powder 2. Elemental analysis: C 73.06% H 11.85% 3. Molecular weight (according to FAB mass spectrum): 738 4. Molecular formula: C ₄₅ H ₈₆ O ₇ 5. Specific rotation: [α] _D ²⁰ = + 5.4 ° (c = 1.0, methanol) 6. Ultraviolet absorption spectrum (in methanol): shows a maximum absorption at 208 nm as shown in FIG. That is, FIG. 1 is a diagram showing an ultraviolet absorption spectrum in the case where this substance is dissolved in methanol at a concentration of 0.25 mg / ml in a relationship between absorbance (vertical axis) and wavelength (nm, horizontal axis). .

7. Infrared external absorption spectrum (KBr tablet): As shown in Fig. 2, 3400, 2990, 2960, 2920, 1380, 1180, 945cm ^-1
Shows absorption. That is, Fig. 2 shows the infrared absorption spectrum of the KBr tablet of this substance in terms of transmittance (%, vertical axis) and wave number (c
m- ¹ (horizontal axis).

8. Color reaction: Ninhydrin reaction is negative, and phenol sulfate reaction does not show sugar color. Positive in iodine reaction and potassium permanganate decolorization reaction.

9. Solubility: Soluble in methanol, acetone, chloroform and ethyl acetate, poorly soluble in water and hexane 10. Distinguishing between acidic, neutral and basic: neutral Known substances that match the above physicochemical properties have been reported. As such, SBS was determined to be a new substance.

(Operation)

Next, the physiological activity of the novel substance SBS of the present invention will be described by experimental examples.

EXPERIMENTAL EXAMPLE 1 Platelet Aggregation Inhibition Collagen having a final concentration of 5 μg / ml was used as a platelet aggregation-causing substance. Japanese white rabbit platelets (4 × 10 ⁵ /
μ) To 0.2 ml, a novel substance SBS having a final concentration of 1 to 50 μg / ml was added, and 30 seconds later, collagen was added, and aggregation of platelets was observed. As a result, as shown in Table 1 below, the novel substance SBS had a clear platelet aggregation inhibitory action.

Experimental Example 2 Anticancer Promoter Action Carcinogenic Promoter Teleocidin B-4 (Teleocidin
B-4) Induced Epstein-Burl (Epstein)
-Barr: The effect of suppressing the early antigen of the virus
ji) Tested in an in vitro system using cells.

Raji cells (5 × 10 ⁵ / ml) were converted to 20 ng / ml teleocidin B
-4, cultivated in a medium containing 5 μm of dimethylsulfoxide and 3 mM n-butyric acid for 48 hours, and cells in which an early antigen was induced were detected by indirect immunofluorescence. Similarly, new substances
As compared with the case where SBS was added at a final concentration of 2 μg / ml, the number of cells that induced the antigen earlier was suppressed to 40% as compared with the case where SBS was not added. As a result, it was revealed that the novel substance SBS has an anti-cancer promoter action.

Experimental Example 3 Acute toxicity test The novel substance of the present invention was orally administered to ICR mice. 1000m
No toxicity was observed at g / kg administration.

As described above, the substance of the present invention, as shown in the above experimental examples,
It exhibits platelet aggregation inhibitory action and anticancer promoter action, and is useful as an antithrombotic agent, an anticancer agent and the like.

The substance also suppresses the blastogenesis of lymphocytes by concanavalin A, and is also useful as an antiallergic agent.

〔Example〕

Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the scope of the following examples.

Example 1 Freeze-dried 1.5 kg of fruiting body
Then, it was pulverized into fine powder with a pulverizer. Add ethyl acetate 3
Extraction was performed for 3 hours while stirring to obtain 5.4 g of an extract. This extraction
Dissolve the product in a small amount of chloroform and use 75 g of silica gel 6
0 (manufactured by Merck) and washed with chloroform
Attached to the ram. After washing with 1 chlorophorom,
Eluted with LUM-methanol (4: 1, V / V) solution.
g was obtained. This eluate is subjected to preparative reversed-phase high-performance liquid chromatography.
Raffy [Column: Capsule Pack C₁₈, φ10 × 250mm, capital
Mobile phase: methanol-water (4: 1, V / V)
Then, the chromatographic pattern shown in FIG. 3 was obtained. That is, the third
The figure shows the chromatographic pattern of reversed-phase high-performance liquid chromatography.
The vertical axis represents absorbance (210 nm), and the horizontal axis represents time.
Indicates the interval (minute).

Each fraction of FIG. 3 was fractionated, A: 18 mg,: B: 39 m
g, C: 44 mg, D: 38 mg, E: 56 mg, F: 61 mg, G: 48 mg and SBS: 1
06 mg was obtained.

Example 2 Liophylum ulmarium Lu1-8 (FERM BP-141
6) 100 ml of seed medium (2% glucose,
Yeast extract 0.3%, peptone 0.5%, KH ₂ PO ₄ 0.1%, MgS
O ₄ · 7H ₂ 0.05% of O) was inoculated into 500ml Erlenmeyer flasks containing, to obtain a seed culture solution was cultured with shaking for 14 days at 25 ° C.. 200 ml of this seed culture was inoculated into a 30-volume jar fermenter containing 10 production media (same composition as the seed medium), and 25
Aeration and agitation culture (aeration 10 / min, agitation 200 rpm) was performed at 6 ° C for 6 days. The cells were collected by a filter press to obtain 830 g of wet cells. After freeze-drying the cells, 49 mg of SBS was obtained in the same manner as in Example 1.

Example 3 The platelet aggregation inhibitory action and the anti-cancer promoter action of each fraction obtained in Example 1 were examined, and the results shown in Table 2 were obtained. The effect of inhibiting platelet aggregation was assayed in an in vitro system using collagen as an aggregating agent.
The assay was performed by suppressing the activation of the bur virus.

(In the table, + represents the intensity of activity, and the intensity is +++++
>++++>++> ++. In addition,-indicates no activity) [Effect of the Invention] The SBS of the present invention is a novel substance produced by the edible bacterium Liophyllum ulmarium and the like, and has a platelet aggregation inhibitory action and an anti-cancer promoter action. ,
It is useful as a functional food material.

[Brief description of the drawings]

Fig. 1 shows the UV absorption spectrum of the novel substance SBS, and Fig. 2 shows the infrared absorption spectrum of the same substance (KBr tablets).
FIG. 3 shows the results of reversed-phase high-performance liquid chromatography of the same substance.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 645) (72) Inventor Morio Hideo 3-4-1 Seta, Otsu-shi, Shiga Pref. 72) Inventor: Hajime Daito 19, Goryo Oiwa-cho, Yamashina-ku, Kyoto, Kyoto (72) Inventor: Koichi Koshimizu 856-10, Horiyamazoe Nishicho, Nara City, Nara Prefecture

Claims (2)

(57) [Claims] 1. A new substance SB having the following physicochemical properties:
S. Elemental analysis: C 73.06% H 11.85% Molecular weight: 738 (by FAB-mass spectrum) Molecular formula: C ₄₅ H ₈₆ O ₇ Ultraviolet absorption spectrum (in methanol): Maximum absorption at 208 nm Red external absorption spectrum (KBr tablet): 3400 , 2990, 2960, 29
20, 1380, 1180, 945cm ^-1 Color reaction: Ninhydrin reaction negative, phenol sulfate reaction negative, iodine reaction positive, potassium permanganate decolorization reaction positive 2. A method for producing a novel substance SBS, which comprises collecting the novel substance SBS according to claim 1 from a culture of a novel substance SBS-producing bacterium belonging to the genus Lyophyllam or a fruiting body of said producing bacterium.